Effective isolation and collection of rare cells from a heterogeneous cell population remains of high interest, due to the increasing demand for isolated cell populations for use in disease diagnosis and treatment, e.g. gene therapy, as well as for basic scientific research. For example, pathologically changed cells, such as cancerous cells, can be separated from a larger normal cell population, and the cleaned cell populations may then be transplanted back into the patient.
One prominent demand is for the isolation of particular fetal cells from heterogeneous maternal cell populations to permit early fetus diagnosis, such as early screening of potential chromosomal disorders during pregnancy. Fetal cells have been obtained by methods such as amniocentesis and chorionic villus sampling; however, such methods can pose risks, especially to the fetus. Some fetal cells are also present in circulating maternal blood, as these cells pass from fetus to the maternal bloodstream in very low numbers; however, the ratio of fetal cells to maternal cells is on the order of only a few ppm. Thus, there are significant challenges associated with the isolation and collection of rare fetal cells from the major population of maternity cells in maternal blood. These challenges also exist in separating fetal cells from cervical mucus, and they may also be common to other rare cell recoveries from bodily fluids or the like, as well as to the separation and isolation of other biomolecules present in only minute quantities.
Cell separation is often achieved by targeting molecules on the cell surface with specific affinity ligands in order to achieve selective, reversible attachment of a target cell population to a solid phase. Nonspecifically adsorbed cells are subsequently removed by washing, and the release of the target cells for analysis may follow. Such specific affinity ligands may be antibodies, lectins, receptors, or other ligands that bind proteins, hormones, carbohydrates, or other such molecules having biological activity.
In addition to column separation, other methods have also now been developed for separating target cells from a diverse population of cells such as may be found in bodily fluid or the like. Published U.S. Patent Application No. 2004/038,315 attaches releasable linkers to the interior luminal surfaces of capillary tubing, with the desired bound cells subsequently being released via a cleavage reagent and recovered. U.S. Published Patent Application No. 2002/132316 uses microchannel devices to separate cell populations through the use of a moving optical gradient field. U.S. Pat. No. 6,074,827 discloses the use of microfluidic devices that are constructed to have “enrichment channels” wherein electrophoresis is used to separate and identify particular nucleic acids from samples. Also mentioned is the optional use of antibodies or other binding fragments to retain a desired target biomaterial. U.S. Pat. No. 6,432,630 discloses a microflow system for guiding the flow of a fluid containing bioparticles through channels where selective deflection is employed, and it indicates that such systems may be used to separate fetal cells from maternal blood samples. The disclosure of these patents and published applications are incorporated herein by reference.
U.S. Pat. No. 6,454,924 discloses microfluidic devices wherein analyte-containing liquids are caused to flow generally downward past sample surfaces disposed atop upstanding pillars on which capture agents are attached, with the side surfaces of such pillars having been rendered hydrophobic so as to facilitate flow in channels that they define.
K. Takahashi et al., in J. Nanobiotechnology, 2, 5 (13 Jun. 2004) (6 pp) (incorporated herein by reference), disclose on-chip cell sorting systems wherein multiple microfluidic inlet passageways lead to a central cell-sorting region fashioned in a PDMS plate (made in a master mold created in photoresist epoxy resin) that is bonded to a glass plate. Agar gel electrodes are provided in the PDMS plate which facilitate the separation of undesired cells by the application of electrostatic forces, that direct these cells into a parallel, continuous stream of buffer, during their flow through a short, cell-sorting region of confluence. A pre-filter which uses posts to physically trap large dust particles is also shown. Published International Application WO 2004/029221 discloses a similarly constructed microfluidic device that can be used for cell separation, such as separating fetal RBCs from maternal blood by selective lysis of maternal RBCs. A sample containing cells may also be introduced into a microfluidic channel device which separates whole cells; it contains a plurality of cylindrical obstacles, with the surfaces of the obstacles having binding moieties, e.g., antibodies, suitably coupled thereto, which moieties will bind to cells in the sample. U.S. Pat. No. 5,637,469 discloses microfluidic devices having a plurality of channels of a depth of 100 microns or less wherein binding moieties, such as antibodies, are immobilized on surfaces to capture biomolecules of interest which can be analyzed in situ. U.S. Pat. No. 5,147,607 teaches the use of devices for carrying out immunoassays, such as sandwich assays, where antibodies are mobilized in microchannels. A recessed area can be provided in the microchannel that contains a group of protrusions which extend upward from the bottom surface of the channel and to which the antibodies are immobilized.
Copending U.S. patent application Ser. Nos. 11/038,920 and 60/678,004, the disclosures of which are incorporated herein by reference, disclose microfluidic devices that can be used for cell separation, such as separating fetal red blood cells from maternal blood or trophoblasts from cervical mucus. A microfluidic channel contains a set irregular pattern of transverse posts which are strategically positioned to disrupt straight-line and streamlined flow and thereby effectively capture target cells with sequestering agents, e.g., antibodies, suitably coupled to the surfaces of the region containing the posts. Although the foregoing briefly described two applications provide improved separation methods for isolating cells or other biomaterials from bodily fluids or the like, this art is considered to be in its infancy, and the search continues for further improvements.